Offspring plumage coloration as a condition‐dependent signal in the blue tit

Abstract In many species, offspring display conspicuous coloration already early in life, even though they might be very vulnerable to predation at this stage. However, most attention has been drawn to the conspicuous plumage displayed by adult individuals in a sexual context, while other signaling functions have been explored much less. Here, we investigated whether the yellow breast plumage of blue tit (Cyanistes caeruleus) nestlings shows patterns of condition dependence and hence signals individual quality, as has been described for adult birds. During three consecutive breeding seasons, we, therefore, explored the association between nestling body mass and three color components of the yellow breast plumage (i.e., UV chroma, carotenoid chroma, and total brightness), considering both within and among nest effects. Variation in carotenoid chroma was not related to body mass. However, UV chroma and total brightness varied with body mass on an among‐nest level, suggesting that they might signal aspects of genetic quality or parental rearing capacity. Interestingly, we also found a within‐nest effect of body mass on total brightness, suggesting that this is a good candidate for a condition‐dependent signal within the family. Thus, other family members could rely on brightness to adjust their behavioral strategies, such as feeding behavior in parents. Our study thus reveals that certain color components of the yellow breast plumage might signal different aspects of offspring quality, and they might have a correlated signaling value across life‐history stages.

discourage potential competitors (Andersson, 1994). Hence, colorful ornaments are thought to function as signals of quality to reliably inform conspecifics about, for example, condition (Hill, 2011), immune status (Rodríguez-Ruiz et al., 2020), or parasitic burden (Megía-Palma et al., 2016). Then, honesty in signaling traits is achieved through associated costs to produce and maintain them (Andersson, 1994).
However, there are conspicuous colorful traits that are also expressed in sexually non-mature or even in newborn individuals. In these cases, coloration is displayed in a non-sexual selection context (West-Eberhard, 1983) at one of the most vulnerable stages in life. These traits have often been explained as by-products of selection acting on colouration in adults, at least when both offspring and adults display the same traits (similar to female ornaments, which, initially, were only interpreted as correlated effects of selection in males; see discussion by Amundsen, 2000; see also Doutrelant et al., 2020;West-Eberhard, 1983). However, given the low-heritability estimates of certain colorations (Charmantier et al., 2017;Class et al., 2019;Drobniak et al., 2013), offspring coloration may have important signaling functions in itself like signaling quality or need to parents. For example, the occurrence of natal coats in primates-distinct from adult fur-seems to have evolved to solicit greater maternal care (Higley et al., 1987). In birds, American coot (Fulica americana) parents preferentially feed the most ornamented offspring (Lyon et al., 1994). Another example is nestling gape coloration, a trait that is adjusted to parent visual performance (Avilés & Soler, 2009) that reflects offspring need (Kilner, 1997; but see Heeb et al., 2003), and that ultimately mediates parental feeding decisions (Gotmark & Ahlstrom, 1997;Kilner, 1997;Saino et al., 2000). There is also some evidence that offspring signals could be perceived by other family members to mediate sib-sib interactions (Dreiss et al., 2016(Dreiss et al., , 2017Roulin et al., 2000).
Yet, while evidence on the adaptive function of offspring ornaments in the context of parental care is accumulating, little is known about how honesty in these signaling traits can be achieved. The expression of structural ornaments such as plumage colouration requires a substantial investment of resources such as carotenoids, and thus, they can inform parents and other family members (such as siblings and breeding helpers) about individual quality (Caro et al., 2016;Hinde & Kilner, 2007;Morales & Velando, 2013). Like in a sexual selection context, honesty can be achieved if the offspring pay a cost for displaying or maintaining such signaling traits, which prevents cheating (handicap principle; Zahavi, 1977). Therefore, nestling coloration has the potential to evolve as a conditiondependent signal to which other family members respond (honest signaling models, Fromhage & Henshaw, 2022;Godfray, 1991Godfray, , 1995Laidre & Johnstone, 2013). Contrastingly, nestlings may also display color traits that function in a sexual context if these are not replaced before sexual maturity. For example, the tail coloration of blue tit nestlings, unlike other plumage patches, is not replaced after the first year (Peters et al., 2007;Svensson, 1992). A good model system to study whether conspicuous nestling plumage coloration shows similar patterns of condition dependence in both offspring and adults is the carotenoid-based coloration of the yellow breast plumage of blue tits (Cyanistes caeruleus). Blue tit adults exhibit both UV/blue crown feathers and yellow breast feathers. In adults, UV/blue coloration might function as a sexual signal (Parker, 2013), as it reflects condition (Delhey et al., 2006) and shapes the parental investment of mates (Limbourg et al., 2013a(Limbourg et al., , 2013b. Similarly, yellow breast feathers reliably reflect aspects of individual quality such as parasite burden (del Cerro et al., 2010), parental capacity (García-Navas et al., 2012), and laying performance (Midamegbe et al., 2013). Furthermore, the UV chroma of adult breast plumage functions as a signal in parental interactions during offspring care (García-Campa et al., 2022). Blue tit nestlings do not exhibit the UV/ blue crown coloration, but there is some evidence that two color parameters of the yellow breast plumage, carotenoid chroma (Johnsen et al., 2003) and UV chroma (Jacot & Kempenaers, 2007;Morales & Velando, 2018), co-vary with nestling body mass. Moreover, family members rely on nestling UV chroma to adjust their decision rules over parental investment. Concretely, nestlings with experimentally blocked UV color beg more during feeding rates and in sib-sib competitive interactions (Morales & Velando, 2018). In addition, when resources are limited, parents favor chicks with higher UV chroma, thus, presumably those of high quality (García-Campa et al., 2021;Morales & Velando, 2018). It is possible that the different components of colouration reveal different aspects of individual quality (Candolin, 2004), as they involve different dimensions of avian color perception (Jacot & Kempenaers, 2007): reflectance in the ultraviolet region of the spectrum (UV chroma; a measure of the contribution of UV to the total reflectance), carotenoid-based reflectance (carotenoid chroma; which reflects the amount of carotenoid pigments deposited in feathers, as it represents the relative reflectance around the absorbance peak of carotenoids), and total reflectance (brightness). Hence, in order to understand the signaling function of yellow breast plumage colouration in blue tit nestlings, it is valuable to investigate the different color components as well as their relationships with condition.
In this study, we first explored the associations between UVchroma, carotenoid chroma, and total brightness of blue tit nestling yellow breast feathers. Then, we investigated the relationship of each of the three color components with body mass in three consecutive breeding seasons. We hypothesized that only individuals in good condition (i.e., nestlings with higher body mass) would be able to achieve, in particular, a higher reflectance in the ultraviolet region of the spectrum, as this has been experimentally demonstrated previously (Morales & Velando, 2018). Furthermore, as multiple chicks per nest were measured, we tested whether any effect of body mass on coloration was due to an among-nest or a within-nest effect, which, to our knowledge, has not been explored to date. The within-nest effect allows testing whether chick coloration varies according to within brood differences in body mass, reflecting condition dependence at the nest level. The among-nest effect in turn would show whether the correlation of body mass and coloration is due to, for instance, genetic effects, parental quality effects, or other (common) environmental effects at the nest level. If nestling yellow plumage functions as a signal in intra-family interactions, we expect a within-nest effect of body mass on coloration, since this would allow other family members to assess individual quality relative to other siblings in the nest. The interaction between the within and the among-nest effect then again would allow testing whether the strength of condition dependence is influenced by brood identity. Unraveling the relationships between offspring colouration and body mass at the among-nest and within-nest level is hence of great importance to better understand the signaling function of coloration.

| General methods
The study was carried out in the locality of Miraflores de la week to record the onset of nest construction. Then, we checked them every 2-3 days to record laying and hatching dates (hatching day = day 0). On days 11 (in 2019) or 12 (in 2017 and 2018), that is, once blue tit nestlings had mostly developed yellow breast feathers (Peters et al., 2007), we measured feather coloration and body mass (see a detailed explanation in Color measurements below). On these days, we also took blood samples (in 2017) and 3-5 breast feathers per nestling for molecular sexing (see below). Blue tit nestlings exhibit yellow breast feathers, which they molt about 2 months after fledging, during the post-juvenile molt (Cramp & Perrins, 1993). Interestingly, blue tit nestlings are sexually dimorphic in the yellow breast feathers at early stages (Johnsen et al., 2003), whereas these differences disappear as adults (Hunt et al., 1998). This pattern is different from other color traits such as the upper-tail feathers, which are sexually dimorphic both in adults (Hunt et al., 1998) and nestlings (Johnsen et al., 2003).

Yellow breast feathers reflect light both in the long-wave band
of the reflectance spectrum (yellow-to-red wavelengths between 550 and 700 nm) and in the ultraviolet (UV) region (Shawkey & Hill, 2005).

| Color measurements
We measured breast plumage coloration with a portable spectrophotometer (Jazz, OceanOptics©) connected to a Pulsed Xenon Light Source (Jazz PX lamp OceanOptics©). For each nestling, we took three consecutive measurements relative to a white standard and perpendicular to the feather surface, using an external probe fitted with a plastic cylinder to standardize the measuring distance and exclude ambient light. We then obtained the reflectance spectra between 320 and 700 nm using CLR program v 1.1 (Montgomerie, 2009). We excluded the first part of the spectrum (300-320 nm) in order to avoid noisy reflectance values.
We then calculated three objective color parameters: (i) total brightness (i.e., average reflectance between 320 and 700 nm; adapted from Jacot & Kempenaers, 2007), (ii) UV chroma (i.e., reflectance in the UV wave-band region of the spectrum divided by the total reflectance of the spectrum in the avian visual range (R 320-400 /R 320-700 ); adapted from Johnsen et al., 2003) and (iii) carotenoid chroma (i.e., an estimation of the carotenoid content of yellow breast feathers (R 700-R 450 /R 700 ), since carotenoids highly absorb in 450 nm; Shawkey & Hill, 2005). For each color parameter, we then calculated the mean of the three consecutive color measurements sampled per nestling.

| Statistical analyses
We used R 4.1.0 (R Core Team, 2020) for statistical analyses. First, to explore how the color parameters were inter-related, we performed correlations between UV chroma, carotenoid chroma, and brightness both at the individual level and at the nest level (the latter using mean values of color parameters). Second, we fitted three linear mixed models with a normal distribution of errors using the lmer function in the "lme4" package (Bates et al., 2015) to determine the relationships between body mass and each of the three color parameters. We assumed normality in all cases after checking the residual plots, given also the robustness of mixed models to violations of normality assumptions (Schielzeth et al., 2020). The models included as fixed effects the average body mass of the brood (=among-nest effect), the deviation from the average body mass of the brood (=within-nest effect), and their interaction. We included in addition year (2017, 2018, and 2019), nestling sex, brood size, and the interactions between year and nestling sex, average body mass and year, average body mass and brood size, and nestling sex with the deviation from the average body mass. Backward elimination for nonsignificant interactions (α = .05) was used to build the minimal models. We also included nest ID as a random intercept and the interaction between nest ID and the deviation from the average body mass (=within-nest effect) as a random slope.

| Associations between color parameters
At the individual level, yellow UV chroma of nestlings was positively correlated with total brightness (r = .35; p < .001; n = 1835) and negatively with carotenoid chroma (r = −.54; p < .001; n = 1835). In contrast, there was no relationship between carotenoid chroma and brightness (r = −.010; p = .70; n = 1835). When analyzing the correlations of these color parameters for each sex separately, the results were consistent.

| Condition dependence
We found a significant among-nest effect of body mass on yellow UV chroma (F 1,223.31 = 8.26; p = .0044; Table 1). Broods in which the average nestling mass was higher had higher levels of UV chroma than broods with on average lower nestling body mass (Figure 2a). However, we did not find a significant within-nest effect of body mass on UV chroma (F 1,189.18 = 2.81; p = .096), so that nestlings with lower than average body mass in their brood did not have lower UV chroma than their siblings (Figure 3a; see also Figure S3). Yellow

UV chroma was significantly affected by the interaction between
year and nestling sex (F 2, 1544.02 = 5.15; p = .0059; Figure S1). UV chroma was higher in females than in males in all the seasons (all Post-Hoc tests: p < .001). In females, UV chroma was higher in 2017 than in the other 2 years, and it did not differ between 2018 and 2019 (p = .23). The same effect was found for males, but, unlike females, the difference between 2018 and 2019 was almost significant (p = .051). The rest of the interactions were not significant (all p > .11).
F I G U R E 1 Correlations between yellow breast feather color parameters (mean nest values) measured in blue tit nestlings: (a) UV chroma versus total brightness, (b) carotenoid chroma versus total brightness, and (c) UV chroma versus carotenoid chroma.
We did not find among-nest (F 1,225.19 = 0.22; p = .64) or within- Interestingly, we found significant both among-nest (F 1,226.76 = 5.22; p = .023) and within-nest effects (F 1,1330.31 = 21.37; p < .001) of body mass on total brightness (Table 1). Thus, broods with higher than average body mass displayed brighter yellow colourations (Figure 2c), and those nestlings with a higher body mass relative to the average body mass of their brood displayed brighter yellow coloration than their siblings (Figure 3c; see also Figure S5).
Total brightness was significantly affected by the interaction between year and nestling sex (F 2,1620.65 = 6.09; p = .0023; Figure S2).
Total brightness was higher for male nestlings than for female nestlings in years 2017 and 2019, while there was not a significant difference in 2018 (Post-Hoc test: p = .28; Figure S2). All other interactions were not significantly different (all p > .30).

| DISCUSS ION
Here, we explored whether the conspicuous coloration of blue tits nestlings could signal quality, by investigating its relationships with body mass both at the within-and the among-nest level. Indeed, as coef. = 0.32 ± 0.10 F 1,1620.65 = 6.09 p = .0023 Note: We included nest ID as a random intercept and the interaction between nest ID and the deviation from the average body mass of the brood (=within-nest effect) as a random slope. Reference levels are "2017" for year effects and "males" for sex effects.
TA B L E 1 Final mixed models exploring the condition-dependence of color parameters. Significant effects are marked in bold.

F I G U R E 2
Among-nest effect of body mass (i.e., average body mass of the brood) on (a) UV chroma, (b) carotenoid chroma, and (c) total brightness of yellow breast feathers measured in blue tit nestlings. Regression lines and ±95% confidence intervals (blue shaded area) are shown.
This pattern was particularly relevant for UV chroma and total brightness. Hence, these traits could act as condition-dependent signals beyond a sexual selection framework, around which most previous work has focused (e.g., del Cerro et al., 2010;Doutrelant et al., 2008Doutrelant et al., , 2012Ferns & Hinsley, 2008;Ferrer et al., 2015;García-Navas et al., 2012;Hidalgo-Garcia, 2006;Midamegbe et al., 2013;Senar et al., 2002). Furthermore, our approach allowed us to explore the potential of the three color components as quality signals both within-nest and among-nest contexts.

| Associations between color parameters
We found a negative association between carotenoid chroma and UV chroma of yellow breast feathers, in line with previous results in other study populations (Johnsen et al., 2003(Johnsen et al., , 2005. This negative association may be due to the fact that higher amounts of carotenoid pigments in the feathers partly conceal feather structures, which results in lower UV reflectance. Moreover, we found that total brightness was strongly and positively associated with UV chroma, but not with carotenoid chroma. Hence, the overall reflectance of yellow breast coloration indicates to a large extent the reflectance in the UV region of the spectrum. This is particularly relevant in our model system, since UV coloration is more easily perceived by cavitynesting birds than carotenoid-based reflectance (Avilés et al., 2006;Hunt et al., 2003;Wȩgrzyn et al., 2011;Wiebe & Slagsvold, 2009).

| Condition dependence: Nestling color as an honest signal of quality
While the color expression of the nestlings' yellow breast feathers showed condition dependency, this effect differed for the three color parameters under study. Furthermore, the contribution of among-nest effects (which encompass a combination of genetic effects, parental quality effects, or other common environmental effects) and within-nest effects (reflecting the relative differences in body mass among all the nestlings raised in the same brood) also varied between the color parameters.
We found a significant among-nest effect of body mass on yellow UV chroma. Broods with higher mean body mass also had higher mean UV chroma. This effect was independent of brood size.
Interestingly, UV chroma could reflect genetic effects (e.g., see UV/ blue chroma: Charmantier et al., 2017), parental rearing capacity (Senar et al., 2002), food stress (Siefferman & Hill, 2005), or parasite infection (del Cerro et al., 2010;Hill, 2006Hill, , 2022, which we cannot separate in our study. However, we did not find differences in UV chroma between nestlings of the same brood (within-nest effect). This suggests that, at the intra-brood level, family members might not use UV chroma as a reliable signal of body mass. This was unexpected since we have experimentally demonstrated in the study population that chicks with reduced UV chroma gain less body mass (Morales & Velando, 2018) and that this trait is used as a signal during intra-family interactions (García-Antón et al., In press;García-Campa et al., 2021;Morales & Velando, 2018). Moreover, cavity-nesting passerines are especially good at detecting changes in UV reflectance (Avilés et al., 2006;Hunt et al., 2003;Wiebe & Slagsvold, 2009), which points to UV chroma as a promising candidate for a signal inside cavities.
However, the current study reveals that total brightness has more potential as a quality signal within nests (as indicated by the significant within-nest effect of body mass) but that it nonetheless reflects UV chroma. Indeed, brightness is an achromatic component brightness, parents could rely on this trait to adjust their feeding strategies within their brood (García-Campa et al., 2021;Mas & Kölliker, 2011;Morales & Velando, 2018). Therefore, total brightness could have evolved as a condition-dependent trait to signal nestling quality to other family members.
Furthermore, the fact that the feathers were collected at different ages from the nestlings (i.e., day 11 in 2019 and day 12 in 2017 and 2018) needs to be discussed. Although breast feathers are almost developed at this age, they are still growing (Peters et al., 2007).

| Differences between the sexes
We also found a consistent effect of nestling sex on the three color parameters analyzed, in accordance with previous studies (Jacot & Kempenaers, 2007;Johnsen et al., 2003Johnsen et al., , 2005. Females expressed higher mean values for yellow UV chroma than males, whereas we detected the opposite pattern for carotenoid chroma and brightness (even though UV chroma and brightness are strongly and positively associated). While the blue tit was one of the first species in which a sexual dimorphism in crown UV-based plumage color was documented, this has not been found in adult yellow breast feathers (Hunt et al., 1998). It is somehow puzzling that the latter trait is dimorphic only in nestlings and juveniles-since yellow body feathers are molted a few months after fledging (Cramp & Perrins, 1993;Schoppe, 1977).
Thus, parents could potentially rely on both carotenoid-chroma and total brightness to discriminate offspring sex while adjusting their feeding strategies. Indeed, in other study populations, blue tit males and females receive different food items (García-Navas et al., 2014) or the total amount of investment (Dickens & Hartley, 2007). In addition, fledging yellow plumage could play a signaling role in family flocks that are formed immediately after fledging (Stenning, 2018) and during social interactions within flocks (Tschirren et al., 2005).
These sex-specific patterns clearly need further study.

| CON CLUS IONS
We show that yellow breast feathers could function as a conditiondependent signaling trait in nestling blue tits, given the observed associations with body mass. While total brightness and UV chroma (but not carotenoid chroma) seem to reflect genetic or other common environmental effects (=among-nest effects), total brightness could also act as an honest signal during intra-family interactions

ACK N OWLED G M ENTS
We would like to thank Sonia González-Braojos and Alicia Rodríguez-Juncá for help during fieldwork and Emilio García Juárez for labwork analyses. We also thank Daniel Díaz and Alejandra Mira (from the Centro de Gestión del Parque Regional de la Cuenca Alta del Río Manzanares) for logistic support. The study was financed by the

CO N FLI C T O F I NTE R E S T S TATE M E NT
Authors declare that they have no conflict of interest. -Campa et al. (2022b).